Lyn, PKC-δ, SHIP-1 interactions regulate GPVI-mediated platelet-dense granule secretion

Abstract

Protein kinase C-␦ (PKC-␦) is expressed in platelets and activated downstream of protease-activated receptors (PARs) and glycoprotein VI (GPVI) receptors. We have previously shown that PKC-␦ positively regulates PAR-mediated dense granule secretion, whereas it negatively regulates GPVI-mediated dense granule secretion. We further investigated the mechanism of such differential regulation of dense granule release by PKC-␦ in platelets. SH2 domain-containing inositol phosphatase-1 (SHIP-1) is phosphorylated on Y1020, a marker for its activation, upon stimulation of human platelets with PAR agonists SFLLRN and AYPGKF or GPVI agonist convulxin. GPVI-mediated SHIP-1 phosphorylation occurred rapidly at 15 seconds, whereas PAR-mediated phosphorylation was delayed, occurring at 1 minute. Lyn and SHIP-1, but not SHIP-2 or Shc, preferentially associated with PKC-␦ on stimulation of platelets with a GPVI agonist, but not with a PAR agonist. In PKC-␦-null murine platelets, convulxin-induced SHIP-1 phosphorylation was inhibited. IntroductionPlatelet-collagen interactions are thought to have the greatest significance at medium or high shear rates found in arteries and diseased vessels. 1 Glycoprotein VI (GPVI) is the major signaling receptor for collagen on the platelet surface. 1 The critical role played by platelets in hemostasis, thrombosis and vascular remodeling, and healing is related to their function as exocytotic cells that secrete important effector molecules at the site of vascular injury. Platelets normally contain at least 3 types of large intracellular granules, such as ␣, dense, and lysosomal granules. There are 3 to 8 dense granules per platelet, and the contents from these dense granules are important for recruiting more platelets to the site of injury. 2,3 Protein kinase C (PKC) has been implicated in platelet secretion. 4 Protein kinase Cs are members of the extended AGC (protein kinases A, G, and C) family of differentially expressed serine/ threonine kinases implicated in a diverse array of cellular functions. After activation, these kinases migrate to different subcellular locations, including the plasma membrane and cytoskeletal elements where they regulate different physiologic functions. 5 PKC isoforms are subdivided into 3 groups based on their lipid and cofactor requirements: the diacylglycerol and calcium-sensitive conventional isoforms (␣, ␤I, ␤II and ␥), the diacylglycerolsensitive and calcium-insensitive novel isoforms (␦, , , and ⑀), and the phosphatidylinositide trisphosphate-sensitive atypical isoforms (, , , and ). 6 PKC-␦ plays a key role in growth regulation and tissue remodeling in other cells 7 and differentially regulates dense granule secretion in platelets. It positively regulates proteaseactivated receptor (PAR)-mediated dense granule release and negatively regulates GPVI-mediated dense granule release. [8][9][10] Intrinsic function of PKCs is regulated by 3 mechanisms: (1) binding of the cofactor that allosterically activates the enzyme, (2) phosphorylation on the activation loop r...

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